Purpose:
Retinal pigment epithelium (RPE), a ciliated monolayer of cells situated adjacent to retinal photoreceptors, is critical for photoreceptor survival and function. Similar to most somatic cells, RPE contains a primary cilium during development. But the role of primary cilium in human RPE development remains largely unknown. The goal of this study is to explore the function and underlying signaling pathway of primary cilium in RPE development and maturation.

Methods:
Human induced pluripotent stem cell derived RPE (iPSC-RPE) were grown on semi-permeable transwells to generate a confluent monolayer. Primary cilia in iPSC-RPE were manipulated using a cilia inducer aphidicolin or cilia function inhibitor HPI-4. Electrophysiology recordings were performed to measure RPE transepithelial potential (TEP) and total tissue resistance (Rt). Immunocytochemistry was used to determine the proper polarization and morphogenesis of iPSC-RPE.

Results:
Six weeks treatment with aphidicolin increased baseline TEP by up to three-folds; it also dramatically enhanced RPE responses to two physiologically relevant stimuli (apical low K+ and ATP). In contrast, suppression of cilium growth with HPI-4 significantly reduced the baseline TEP/Rt and blocked the low K+ and ATP responses, suggesting that cilium function is crucial for the polarization and maturation of RPE monolayer. Pharmacological inhibition of PKC-δ, a polarization inducing kinase, with rottlerin attenuated the effect of aphidicolin in a dose-dependent manner; similar inhibitory effects were also observed with another PKC-δ blocker Go6850. Positive immunostaining for the active phosphorylated form of PKC-δ and Phospho-MLC further confirmed that aphidicolin-induced polarization in the RPE monolayer acted through PKC-δ pathway.

Conclusions:
These results indicate that the development of primary cilium in iPSC-RPE has a direct and dramatic impact on RPE monolayer polarization and maturation through modulation of PKC-δ pathway. Manipulation of this pathway can help differentiate and mature RPE cells for stem cell-based therapies.